title: Cloning and functional characterization of endo-β-1,4-glucanase gene from metagenomic library of vermicompost.
abstract: In the vermicomposting of paper mill sludge, the activity of earthworms is very dependent on dietetic polysaccharides including cellulose as energy sources. Most of these polymers are degraded by the host microbiota and considered potentially important source for cellulolytic enzymes. In the present study, a metagenomic library was constructed from vermicompost (VC) prepared with paper mill sludge and dairy sludge (fresh sludge, FS) and functionally screened for cellulolytic activities. Eighteen cellulase expressing clones were isolated from about 89,000 fosmid clones libraries. A short fragment library was constructed from the most active positive clone (cMGL504) and one open reading frame (ORF) of 1,092 bp encoding an endo-β-1,4-glucanase was indentified which showed 88% similarity with Cellvibrio mixtus cellulase A gene. The endo-β-1,4-glucanase cmgl504 gene was overexpressed in Escherichia coli. The purified recombinant cmgl504 cellulase displayed activities at a broad range of temperature (25-55°C) and pH (5.5-8.5). The enzyme degraded carboxymethyl cellulose (CMC) with 15.4 U, while having low activity against avicel. No detectable activity was found for xylan and laminarin. The enzyme activity was stimulated by potassium chloride. The deduced protein and three-dimensional structure of metagenome-derived cellulase cmgl504 possessed all features, including general architecture, signature motifs, and N-terminal signal peptide, followed by the catalytic domain of cellulase belonging to glycosyl hydrolase family 5 (GHF5). The cellulases cloned in this work may play important roles in the degradation of celluloses in vermicomposting process and could be exploited for industrial application in future.
authors.affiliation: King Fahd Medical Research Center, King Abdulaziz University, Jeddah 21589, Saudi Arabia.
authors.forename: Muhammad
authors.initials: M
authors.lastname: Yasir
authors.forename: Haji
authors.initials: H
authors.lastname: Khan
authors.forename: Syed Sikander
authors.initials: SS
authors.lastname: Azam
authors.forename: Amar
authors.initials: A
authors.lastname: Telke
authors.forename: Seon Won
authors.initials: SW
authors.lastname: Kim
authors.forename: Young Ryun
authors.initials: YR
authors.lastname: Chung
mesh_terms: D002266:Carboxymethylcellulose Sodium / Q000378:metabolism; D002480:Cellulase / Q000235:genetics / Q000378:metabolism*; D002482:Cellulose / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D056186:Metagenomics / Q000379:methods*; D016366:Open Reading Frames / Q000235:genetics
chemical_list: D002482:Cellulose; D002480:Cellulase; D002266:Carboxymethylcellulose Sodium